Methods and arrays for target analyte detection and determination of reaction components that affect a reaction

ABSTRACT

Methods are described for detecting reaction components with affect a reaction. Biomolecules such as enzymes can be addressed at the single molecule level in order to discover function, detect binding partners or inhibitors, and/or measure rate constants.

GOVERNMENT RIGHTS

The United States government may have certain rights in this inventionpursuant to Contract No. N00014-01-1-0659 awarded by the Department ofDefense, Defense Advanced Research Projects Agency (DARPA) Office ofNaval Research.

BACKGROUND

Methods that implement high-sensitivity and low-level analyte detectionin conjunction with rapid and reproducible experimental protocols arethe cornerstone of modern analytical measurements. Currently, most knowntechniques for quantifying low levels of target analyte in a samplematrix use amplification procedures to increase the number of reportermolecules and thereby provide a measurable signal. These known processesinclude enzyme-linked immunosorbent assays (ELISA) for amplifying thesignal in antibody-based assays, as well as the polymerase chainreaction (PCR) for amplifying target DNA strands in DNA-based assays. Amore sensitive but indirect protein target amplification technique,called immuno-PCR (see Sano, T.; Smith, C. L.; Cantor, C. R. Science1992, 258, 120-122), makes use of oligonucleotide markers, which cansubsequently be amplified using PCR and detected using a DNA assay (seeNam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884-1886;Niemeyer, C. M.; Adler, M.; Pignataro, B.; Lenhert, S.; Gao, S.; Chi, L.F.; Fuchs, H.; Blohm, D. Nucleic Acids Research 1999, 27, 4553-4561; andZhou, H.; Fisher, R. J.; Papas, T. S. Nucleic Acids Research 1993, 21,6038-6039). While the immuno-PCR method permits ultra low-level proteindetection, it is a complex assay procedure, and can be prone tofalse-positive signal generation (see Niemeyer, C. M.; Adler, M.;Wacker, R. Trends in Biotechnology 2005, 23, 208-216).

One disadvantage of these known methods is their reliance on separatesteps to amplify reporter molecules to provide a measurable signal,thereby requiring additional amplification steps and thus additionaltime, equipment, and materials.

In addition, known methods for accurately quantifying the concentrationof a particular analyte in solution are all based on ensemble responsesin which many analyte molecules give rise to the measured signal.

Therefore, there is a need in the art for an improved method and systemof target analyte detection.

BRIEF SUMMARY OF THE INVENTION

According to one embodiment, the present invention relates to a methodof detecting a target analyte in a sample. The method includes providingan array comprising a plurality of sites, each site comprising a capturecomponent, and contacting the array with the sample such that each sitein a subset of the plurality of sites contains a single target analyte.Each target analyte comprises an enzymatic component. The method furtherincludes contacting the array with an enzymatic substrate and detectinga change in an optical property at each of the sites as an indication ofthe presence of the target analyte.

The present invention, in another embodiment, relates to a method ofdetecting target analytes in a sample. The method includes providing anarray comprising a plurality of sites, and contacting the array with thesample such that each site in a first subset of the plurality of sitescontains a single first target analyte and each site in a second subsetof the plurality of sites contains a single second target analyte. Inthis embodiment, each site comprises a capture component and each of thefirst and second target analytes comprises an enzymatic component. Themethod further includes contacting the array with a first enzymaticsubstrate and detecting any change in an optical property as a result ofthe first enzymatic substrate at each of the sites as an indication ofthe presence of one of the first or second target analytes. In addition,the method includes washing the array and contacting the array with asecond enzymatic substrate. Further, the method includes detecting anychange in an optical property as a result of the second enzymaticsubstrate at each of the sites as an indication of the presence of oneof the first or second target analytes.

In accordance with another embodiment, the present invention relates toa method of detecting a target analyte in a sample. The method includesproviding an array comprising a plurality of sites and contacting thearray with the sample such that each site in a subset of the pluralityof sites contains a single target analyte. In this method, each sitecomprises a capture component. The method also includes contacting eachof the single target analytes with a binding ligand comprising anenzymatic component and further contacting the array with an enzymaticsubstrate. In addition, the method includes detecting a change in anoptical property at each of the sites as an indication of the presenceof the target analyte.

The present invention, according to a further embodiment, is a method ofquantifying an amount of a target analyte in a sample. The methodincludes providing an array comprising a plurality of sites, each sitecomprising a capture component and contacting the array with the samplesuch that each site in a subset of the plurality of sites contains asingle target analyte. In this embodiment, each target analyte comprisesan enzymatic component. The method also includes contacting the arraywith an enzymatic substrate, detecting a change in an optical propertyat each of the sites as an indication of the presence of the targetanalyte, and calculating an amount of the target analyte in the sample.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. As will be realized, theinvention is capable of modifications in various obvious aspects, allwithout departing from the spirit and scope of the present invention.Accordingly, the drawings and detailed description are to be regarded asillustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a, 1 b, and 1 c are side view cross-section schematicsrepresenting etched bundle modifications, according to one embodiment ofthe present invention.

FIGS. 2 a, 2 b, and 2 c are side view cross-section schematicsrepresenting a sandwich assay, according to one embodiment of thepresent invention.

FIGS. 3 a and 3 b are photographs depicting Streptavidin Alexa Fluor568® binding to (a) an unpolished biotin modified fiber optic array, and(b) a polished biotin modified fiber optic array, according to oneembodiment of the present invention.

FIGS. 4 a, 4 b, 4 c, 4 d, 4 e, and 4 f are photographs depictingexperiments according to one embodiment of the present invention inwhich β-galactosidase hydrolyzes RDG to form resorufin. Morespecifically, each of these figures depicts a different sample having adifferent concentration of SβG. The concentrations were: (a) 128 amol,(b) 51 amol, (c) 25 amol, (d) 7.5 amol, and (e) 2.6 amol, and (f) wasthe control.

FIG. 5 is a chart depicting a log-log pit of the moles of target presentin a sample with the resulting percentage of active reaction vessels,according to one embodiment of the present invention.

FIG. 6 a is a microscopic photograph of an entire fiber array and aninset close-up of the bundle, according to one embodiment of the presentinvention.

FIG. 6 b is an AFM image of a portion of an etched surface, according toone embodiment of the present invention.

FIGS. 7 a, 7 b, and 7 c depict enclosure of the reaction vessels andevaluation of the seal, according to one embodiment. FIG. 7 a is amicroscopic photograph of a solution of Ru(bpy)₃Cl₂ enclosed in thearray of chambers. FIG. 7 b is a microscopic photograph of a smalloctagonal portion of the bundle photobleached with UV light. FIG. 7 c isa microscopic photograph of FIG. 7 b taken 60 minutes later.

FIGS. 8 a, 8 b, and 8 c are microscopic photographs depicting detectionof the activity of single molecules of β-galactosidase, according tovarious embodiments of the present invention. FIG. 8 a is a microscopicphotograph of a background image of a portion of an array. FIG. 8 b is amicroscopic photograph of an image taken of a portion of a 1:5 enzyme tovessel assay. FIG. 8 c is a microscopic photograph of a 1:80 enzyme tovessel assay.

DETAILED DESCRIPTION

The present invention relates to methods, systems, and devices forenzymatic detection and quantification of a target analyte or targetanalytes in a sample. More specifically, the present invention relatesto enzymatic detection and quantification of target analytes usingarrays of micron- to nanoscale-sized reaction vessels containing capturecomponents. According to one embodiment, an array of reaction vesselscontaining capture components is contacted with a sample containing atleast one target analyte. A chromogenic substrate is then added and theresulting chromogenic product of the enzymatic reaction allows fordetection of the analyte. Further, according to one embodiment, thepercentage of reaction vessels with captured target analytes can be usedto calculate the amount of target analyte in the sample using a binaryreadout method.

More specifically, the present invention provides for an array ofmicron- to nanoscale-sized reaction vessels specifically functionalizedand capable of capturing target molecules that are enzymes orenzyme-labelled. The ability to immobilize the target allows the use ofwashing steps and indirect assays, as outlined below. In use, singleenzyme (or enzyme-labelled) molecules are captured in individualreaction vessels and catalyze the production of a sufficient number ofchromogenic product molecules to generate a detectable signal. Inaccordance with one embodiment relating to samples having low targetanalyte concentrations, only a portion of the reaction vessels bind atarget molecule, thereby enabling a binary readout of targetconcentration from the array.

Thus, the direct enzymatic amplification in the method and system of thepresent invention allows for direct amplification of a detectablesignal. Further, unlike the prior art methods, the present inventionallows for detection of low concentrations of protein.

The quantification method, according to one embodiment, is a novelmethod for concentration determination based on statistical analysis.The sample enzyme concentration is determined by distributing theenzyme-containing sample and a suitable substrate, into many nanoscalereaction vessels. In this method, the vessels contain either zero or oneenzyme molecule. By observing the presence or absence of a fluorescentproduct resulting from single enzyme molecule catalysis in each reactionvessel, a binary readout method can be used to count enzyme molecules.Finally, the percentage of reaction vessels occupied by enzyme moleculesis correlated to the bulk enzyme concentration.

I. Arrays

The present invention provides array compositions comprising at least afirst substrate with a surface comprising a plurality of assaylocations. By “array” herein is meant a plurality of capture componentsin an array format. The size of the array will depend on the compositionand end use of the array. Arrays containing from about 2 differentcapture components to many millions can be made, with very large arraysbeing possible, including very large fiber optic arrays. Generally, thearray will comprise from two to as many as a billion or more capturecomponents, depending on the size of the wells and the substrate, aswell as the end use of the array, thus very high density, high density,moderate density, low density and very low density arrays may be made.Preferred ranges for very high density arrays are from about 10,000,000to about 2,000,000,000, with from about 100,000,000 to about1,000,000,000 being preferred. High density arrays range about 100,000to about 10,000,000, with from about 1,000,000 to about 5,000,000 beingparticularly preferred. Moderate density arrays range from about 10,000to about 50,000 being particularly preferred, and from about 20,000 toabout 30,000 being especially preferred. Low density arrays aregenerally less than 10,000, with from about 1,000 to about 5,000 beingpreferred. Very low density arrays are less than 1,000, with from about10 to about 1000 being preferred, and from about 100 to about 500 beingparticularly preferred. In some embodiments, multiple substrates may beused, either of different or identical compositions. Thus for example,large arrays may comprise a plurality of smaller substrates.

The compositions comprise a substrate. By “substrate”, “array substrate”or “solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of target analytes and isamenable to at least one detection method. As will be appreciated bythose in the art, the number of possible substrates are very large, andinclude, but are not limited to, glass and modified or functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose,composite materials, ceramics, and plastic resins, silica orsilica-based materials including silicon and modified silicon, carbon,metals, inorganic glasses, plastics, optical fiber bundles, and avariety of other polymers. In general, the substrates allow opticaldetection and do not appreciably fluoresce.

In one embodiment, the substrate comprises the end of an optical fiberbundle. Alternatively, the substrate does not comprise the ends of anoptical fiber bundle. For example, the substrate may be a spotted,printed or photolithographic substrate known in the art; see for exampleWO 95/25116; WO 95/35505; PCT US98/09163; U.S. Pat. Nos. 5,700,637;5,807,522 and 5,445,934; and U.S. Ser. Nos. 08/851,203 and 09/187,289,and references cited within, all of which are expressly incorporated byreference. One advantage of using the distal end of a optical fiberbundle as a substrate in the present invention is that the individualfibers in contact with each well can be used to carry both excitationand emission light to and from the wells, enabling remote interrogationof the well contents. Further, an array of optical fibers provides thecapability for simultaneous excitation of molecules in adjacent vessels,without signal “cross-talk” between fibers. That is, excitation lighttransmitted in one fiber does not escape to a neighboring fiber.

In one embodiment, the substrate is planar, although as will beappreciated by those in the art, other configurations of substrates maybe used as well; for example, three dimensional configurations can beused. Preferred substrates include optical fiber bundles as discussedbelow, and flat planar substrates such as glass, polystyrene and otherplastics and acrylics.

In one embodiment, at least one surface of the substrate is modified tocontain discrete, individual sites (also referred to herein as “reactionvessels” and “microwells”) for later association of target analytes.These sites generally comprise physically altered sites, i.e. physicalconfigurations such as wells or small depressions in the substrate thatcan retain the beads. The microwells may be formed as is generally knownin the art using a variety of techniques, including, but not limited to,photolithography, stamping techniques, molding techniques andmicroetching techniques. As will be appreciated by those in the art, thetechnique used will depend on the composition and shape of thesubstrate.

In one embodiment, physical alterations are made in a surface of thesubstrate to produce the sites. In a preferred embodiment, the substrateis a fiber optic bundle and the surface of the substrate is a terminalend of the fiber bundle. In this embodiment, wells are made in aterminal or distal end of a fiber optic bundle comprising individualfibers. In this embodiment, the cores of the individual fibers areetched, with respect to the cladding, such that small wells ordepressions are formed at one end of the fibers. The required depth ofthe wells will depend on the size of the beads to be added to the wells.In one aspect of the present invention, the physical alterations can bemade as taught in U.S. Pat. Nos. 6,023,540, 6,327,410, and 6,858,394,which are each incorporated by reference herein in their entirety.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of beads on the substrate.

In accordance with one embodiment of the present invention, the reactionvessels have a volume ranging from about 10 attoliters to about 50picoliters. Alternatively, the reaction vessels range in size from about1 femtoliter to about 1 picoliter. In a further alternative, thereaction vessels range from about 30 femtoliters to about 60femtoliters.

In one aspect of the present invention, the array is a fiber opticarray. The array, according to one embodiment, can be made as follows.First, the reaction vessels are formed on the distal end of a fiberoptic bundle. According to one embodiment, the vessels are created usingan etching process, such as, for example, an acid etching process,resulting in reaction vessels of the desired volume. That is, theetching process creates depressions or holes in the core material at theend of the fiber bundle, while the cladding material is not impacted,thus resulting in reaction vessels. Alternatively, both the corematerial and cladding material are etched, but the cladding material isetched at a slower rate than the core material, thereby resulting inreaction vessels. One advantage of the fiber optic array format is thatit circumvents a complicated microfabrication procedure and provides theability to observe many reaction vessels simultaneously.

II. Capture Components

The microwells of the present invention comprise at least one capturecomponent. A capture component (also referred to as a “capture bindingligand,” “binding ligand,” “capture binding species,” or “captureprobe”) is any molecule, compound, or microwell modification that can beused to probe for, attach, bind or otherwise capture a target analytewithin a microwell on the substrate, such that the target analyte isimmobilized during the assay. Generally, the capture binding ligand orcomponent allows the attachment of a target analyte to the microwell,for the purposes of detection, quantification, or other analysis.

As will be appreciated by those in the art, the composition of thecapture component will depend on the composition of the target analyte.Capture components for a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is aprotein, the capture components or binding ligands include proteins(particularly including antibodies or fragments thereof (FAbs, etc.)) orsmall molecules. Preferred capture component proteins include peptides.For example, when the analyte is an enzyme, suitable binding ligandsinclude substrates and inhibitors. Antigen-antibody pairs,receptor-ligands, and carbohydrates and their binding partners are alsosuitable analyte-binding ligand pairs. In addition, when the analyte isa single-stranded nucleic acid, the binding ligand may be acomplementary nucleic acid. Similarly, the analyte may be a nucleic acidbinding protein and the capture binding ligand is either single-strandedor double stranded nucleic acid; alternatively, the binding ligand maybe a nucleic acid-binding protein when the analyte is a single ordouble-stranded nucleic acid. Alternatively, as is generally describedin U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459,5,683,867, 5,705,337, and related patents, hereby incorporated byreference, nucleic acid “aptomers” can be developed for binding tovirtually any target analyte. As will be appreciated by those in theart, any two molecules that will associate may be used, either as ananalyte or as the capture component. Similarly, there is a wide body ofliterature relating to the development of capture components based oncombinatorial chemistry methods.

Suitable analyte/capture component pairs include, but are not limitedto, antibodies/antigens, receptors/ligands, proteins/nucleic acid,enzymes/substrates and/or inhibitors, carbohydrates (includingglycoproteins and glycolipids)/lectins, proteins/proteins,proteins/small molecules; and carbohydrates and their binding partnersare also suitable analyte-binding ligand pairs. These may be wild-typeor derivative sequences. According to one embodiment, the capturecomponents are portions (particularly the extracellular portions) ofcell surface receptors that are known to multimerize, such as the growthhormone receptor, glucose transporters (particularly GLUT 4 receptor),transferring receptor, epidermal growth factor receptor, low densitylipoprotein receptor, high density lipoprotein receptor, epidermalgrowth factor receptor, leptin receptor, interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGFreceptor, PDGF receptor, EPO receptor, TPO receptor, ciliaryneurotrophic factor receptor, prolactin receptor, and T-cell receptors.

In a preferred embodiment, the capture component is attached to themicrowell or reaction vessel as outlined herein, for example via an“attachment component” (also referred to herein as an “attachmentlinker”). An “attachment component,” as used herein, is defined as anycomponent, functionalization, or modification of the microwells thatresults in the attachment of the capture component, and can includebonds and/or linkers. Alternatively, the capture component may utilize acapture extender component. In this embodiment, the capture component orbinding ligand comprises a first portion that will bind the targetanalyte and a second portion that can be used for attachment to thesurface.

The method of attachment of the capture binding ligand to the attachmentlinker will generally be done as is known in the art, and will depend onthe composition of the attachment linker and the capture binding ligand.In general, the capture binding ligands are attached to the attachmentlinker through the use of functional groups on each that can then beused for attachment. According to one embodiment, the functional groupis a chemical functionality. That is, the microwell surface isderivatized such that a chemical functionality is bound to the surface.Preferred functional groups for attachment are amino groups, carboxygroups, oxo groups and thiol groups. These functional groups can then beattached, either directly or through the use of a linker, sometimesreferred to herein as a “cross-linker.” Linkers are known in the art;for example, homo- or hetero-bifunctional linkers as are well known (see1994 Pierce Chemical. Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).Preferred linkers include, but are not limited to, alkyl groups(including substituted alkyl groups and alkyl groups containingheteroatom moieties), with short alkyl groups, esters, amide, amine,epoxy groups and ethylene glycol and derivatives being preferred.Linkers may also be a sulfone group, forming sulfonamide.

According to one embodiment, the functional group is a light-activatedfunctional group. That is, the functional group can be activated bylight to attach to the target analyte or to the crosslinker. One exampleis the PhotoLink™ technology available from SurModics, Inc. in EdenPrairie, Minn.

In one alternative aspect of the invention, the functional group isadded without derivatizing the well surface. That is, the functionalgroups can be added to the surface by adding a molecule having anattached functional group attached, wherein the molecule has a bindingaffinity for the well surface. The molecule, according to one embodimentis bovine serum albumin. Alternatively, the molecule is any proteincapable of binding or sticking to the vessel surface. In a furtheralternative, the molecule is any molecule capable of binding or stickingto the vessel surface. In one example, the molecule is bovine serumalbumin with free amine groups on its surface. The crosslinker can thenbe added to attach to the amine groups.

According to one exemplary embodiment in which the capture component isa chemical crosslinker, the target analyte is attached using chemicalcrosslinking in the following manner. First, the reaction vessel surfaceis derivatized with a functional group such as NH₂. Next, thecrosslinker and the target analyte are added to the array such that thecrosslinker attaches to the NH₂ and the target analyte attaches to thecrosslinker. In an alternative embodiment described in further detailbelow in which the target analyte is not an enzyme, a label having anenzymatic component can also be attached to the target analyte.

In this way, capture binding ligands comprising proteins, lectins,nucleic acids, small organic molecules, carbohydrates, etc. can beadded.

One embodiment utilizes proteinaceous capture components or capturebinding ligands. As is known in the art, any number of techniques may beused to attach a proteinaceous capture binding ligand. “Protein” in thiscontext includes proteins, polypeptides, peptides, including, forexample, enzymes. A wide variety of techniques are known to add moietiesto proteins. One preferred method is outlined in U.S. Pat. No.5,620,850, hereby incorporated by reference in its entirety. Theattachment of proteins to surfaces is known; see also Heller, Acc. Chem.Res. 23:128 (1990), and related work.

An alternative embodiment utilizes nucleic acids as the capture bindingligand, for, example for when the target analyte is a nucleic acid or anucleic acid binding protein, or when the nucleic acid serves as anaptamer for binding a protein, as is well known in the art.

According to one embodiment, each microwell comprises a plurality ofcapture components. The plurality of capture components, in one aspectof the invention, are distributed on the surface of the well like a“lawn.” Alternatively, the capture components are distributed in anyknown fashion.

The binding between the capture component and the target analyte, inaccordance with one embodiment, is specific and the capture component ispart of a binding pair. That is, the capture component is a targetspecific capture component that specifically binds with or hasspecificity for the target analyte. More specifically, the capturecomponent binds specifically and directly to the target analyte. By“specifically bind” or “binding specificity” herein is meant that thecapture component binds the analyte with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. For example, the capture component according to oneembodiment is an antibody that binds specifically to some portion of thetarget analyte. The antibody, according to one embodiment, can be anyantibody capable of binding specifically to a target analyte. Forexample, appropriate antibodies include, but are not limited to,monoclonal antibodies, bispecific antibodies, minibodies, domainantibodies, synthetic antibodies (sometimes referred to as “antibodymimetics”), chimeric antibodies, humanized antibodies, antibody fusions(sometimes referred to as “antibody conjugates”), and fragments of each,respectively.

However, as will be appreciated by those in the art, it is possible todetect analytes using binding which is not highly specific; for example,the systems may use different capture components such as, for example,an array of different ligands, and detection of any particular analyteis via its “signature” of binding to a panel of binding ligands, similarto the manner in which “electronic noses” work. This finds particularutility in the detection of chemical analytes. The binding should besufficient to remain bound under the conditions of the assay, includingwash steps to remove non-specific binding. In some embodiments, forexample in the detection of certain biomolecules, the binding constantsof the analyte to the binding ligand will be at least about 10⁴-10⁶ M⁻¹,with at least about 10⁵ to 10⁹ M⁻¹ being preferred and at least about10⁷-10⁹ M⁻¹ being particularly preferred.

According to one embodiment in which the target analyte is a cell,including, for example, bacterial cells, the capture component is anadhesin receptor molecule. In use, the adhesin receptor molecule bindswith a surface protein called an adhesin on the extracellular surface ofthe target cell, thereby immobilizing or capturing the cell.Alternatively, in embodiments in which the target analyte is anothertype of cell (a non-bacterial cell), the capture component is anappropriate cell surface receptor that binds the target analyte cell. Ina further embodiment in which the target analyte is a cell, the capturecomponent is fibronectin. For example, fibronectin can be used when thetarget analyte is a nerve cell.

Alternatively, the capture component is a non-specific capturecomponent. That is, the capture component does not bind specifically toa target analyte, but rather binds to a corresponding binding partnerassociated with or attached to the target analyte. For example, thenon-specific capture component according to one embodiment is a chemicalcross-linker as described above. According to one embodiment, everypeptide molecule in a target sample can attach to the chemicalcross-linker. This type of system can be used to identify enzyme targetanalytes because the analytes are detected by modifying the substrate.

In one example of a non-specific capture component according to oneembodiment, the capture component is streptavidin, which binds with highaffinity to biotin, and thus binds to any molecule to which biotin hasbeen attached. Alternatively, the capture component is biotin, andstreptavidin is attached to or associated with the target analyte suchthat the target analyte can be captured by the biotin.

According to one embodiment, the capture component is added to thereaction vessels in the following manner. First, the microwells areprepared for attachment of the capture component(s). That is, themicrowells are modified or an attachment component is added to themicrowells such that the capture component(s) will attach to themicrowells. In one embodiment, the microwells are derivatized with achemical functionality as described above. Next, the capture componentis added.

One example of capture component attachment is depicted in FIG. 1, inwhich reaction vessels of the present invention are functionalized withbiotin. As shown in FIG. 1 a, the array of the present invention in thisexample is a fiber optic bundle 10. To attach the capture component 18,the microwells are first modified with an attachment component 16, whichin this example is an aminopropyl silane 16 that is bound to both thecore 12 and cladding 14 surfaces of the distal end of the fiber bundle10, as shown in FIG. 1 b. The modification with aminopropyl silane iseffective in this example because NHS-biotin attaches to anamino-silanized surface 16. However, since the capture component 18should be present only within the reaction vessels, the externalsurfaces of the substrate, such as the external surfaces of the cladding14, should not be silanized. That is, the silanization must be removedfrom the external cladding surface 14 to avoid biotin attachment. Inthis example as shown in FIG. 1 c, the silanization 16 was removed fromthe external cladding layer 14 by polishing the amino-silanized fibersfor 10 seconds with 0.3 μm lapping film, thereby removing the topamino-silanized cladding layer.

After the attachment component 16 has been added to the microwells, thecapture component 18 can be attached. In the example in FIG. 1, thecapture component 18 is biotin 18. As shown in FIG. 1 d, biotinsuccinimidyl ester 18 is attached to the amino groups 16 on the wellsurfaces 12.

III. Target Analytes

As discussed herein, the array of the present invention provides fordetection, quantification, and further analysis of target analytes. By“target analyte” or “analyte” or grammatical equivalents herein is meantany atom, molecule, ion, molecular ion, compound or particle to beeither detected or evaluated for binding partners.

According to one embodiment, the target analyte is an enzyme. Forexample, the enzyme can be an enzyme from any of the six enzymeclassifications: oxidoreductases, transferases, hydrolases, lyases,isomerases, and ligases. Thus, appropriate enzymes include, but are notlimited to, polymerases, cathepsins, calpains, amino-transferases suchas, for example, AST and ALT, proteases such as, for example, caspases,nucleotide cyclases, transferases, lipases, enzymes associated withheart attacks, and the like. When the system of the present invention isused to detect viral or bacterial targets, appropriate enzymes includeviral or bacterial polymerases and other such enzymes, including viralor bacterial proteases.

Alternatively, the target analyte has an enzymatic component. Forexample, the target analyte can be a cell having an enzyme or enzymaticcomponent present on its extracellular surface. Alternatively, thetarget analyte is a cell having no enzymatic component. Such a cell istypically identified using an indirect assaying method described belowsuch as a “sandwich” assay.

In accordance with another embodiment, the target analyte is not anenzyme. As will be appreciated by those in the art, a large number ofanalytes may be used in the present invention; basically, any targetanalyte can be used which binds a capture component and/or a secondarybinding ligand. As will be explained in further detail below, thesetarget analytes are typically identified using an indirect assay such asa “sandwich” assay. As mentioned above, one suitable target analyte is acell. In addition, suitable analytes include organic and inorganicmolecules, including biomolecules. In a preferred embodiment, the targetanalyte is a protein. As will be appreciated by those in the art, thereare a large number of possible proteinaceous target analytes that may bedetected or evaluated for binding partners using the present invention.In addition to enzymes as discussed above, suitable protein targetanalytes include, but are not limited to, (1) immunoglobulins; (2)hormones and cytokines (many of which serve as ligands for cellularreceptors); and (3) other proteins.

According to one embodiment in which the target analyte is not an enzymeand a sandwich assay is performed as described in further detail below,the enzymatic label as described in further detail below can be betagalactosidase. Alternatively, the enzyme label can be, but is notlimited to, alkaline phosphatase or horseradish peroxidase.

Further suitable target analytes include, but are not limited to, anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, nucleic acids, lipids, carbohydrates, cellular membraneantigens and receptors (neural, hormonal, nutrient, and cell surfacereceptors) or their ligands, etc); whole cells (including procaryotic(such as pathogenic bacteria) and eukaryotic cells, including mammaliantumor cells); viruses (including retroviruses, herpesviruses,adenoviruses, lentiviruses, etc.); and spores; etc.

IV. Enzymatic Substrate

After the target analyte(s) are captured within the microwell(s) (andafter a washing step, according to certain embodiments), a reactioncomponent is added to the array. By “reaction component,” as usedherein, is meant a molecule that affects an enzymatic reaction whencontacted with an enzyme or enzymatic molecule. By “affects” a reactionis meant to include, but is not limited to, inducing, activating, oraltering (for example, slowing down or speeding up) a reaction, orinhibiting a reaction. According to one embodiment, the reactioncomponent is a chromogenic enzymatic substrate. A “chromogenic enzymaticsubstrate” as used herein is any molecule that is converted by an enzymeinto a chromogenic product as a result of an enzymatic reaction.“Chromogenic” means relating to color or pigment in the optical (visiblelight) spectrum and includes fluorogenic.

It is understood in the art that chromogenic substrates are known or canbe made for enzymes in any of the six enzyme classifications. Thus, anyknown chromogenic substrate capable of producing a chromogenic productin a reaction with a particular enzyme can be used in the presentinvention, including any of the chromogenic enzyme substrates disclosedin The Handbook—A Guide to Fluorescent Probes and Labeling Technologies,Tenth Ed., Chapter 10,http://probes.invitrogen.com/handbook/sections/1000.html, which isincorporated herein by reference in its entirety.

According to one embodiment in which the assay of the present inventionis a sandwich assay as described further herein in which the enzymelabel is beta galactosidase, the substrate added to the array is a betagalactosidase substrate such as resorufin-β-D-galactopyranoside.

V. Assay Methods

The array of the present invention can be used for several differentassay methods. More specifically, the present invention provides forboth (a) target analyte detection and (b) quantification of targetanalyte concentration in a sample.

Generally, the system or array of the present invention is exposed to ananalyte of interest (or contacted with a sample containing an analyte ofinterest) and the analyte is immobilized by a capture component in amicrowell, under conditions suitable for immobilization of the targetanalyte to at least one of the capture components, i.e. generallyphysiological conditions. For purposes of the present application, theterm “immobilized” means attached, bound, or affixed to a capturecomponent in a microwell. Thus, the interaction between any analytemolecule and the capture component in a microwell results inimmobilization of the analyte molecule within that microwell.

According to one aspect of the invention, the sample of interest isplaced in contact with the array of the present invention (or the arrayis incubated in the sample) for a period of from about 45 minutes toabout 75 minutes. Alternatively, the array and sample are contacted fora period of from about 50 minutes to about 70 minutes. In a furtheralternative, the incubation period is about 1 hour.

According to one embodiment, a wash step is performed after contactingthe array with the sample. The wash step is intended to wash away anytarget analytes or non-target molecules that are not bound to a capturecomponent. Alternatively, no wash step is needed.

In one aspect of the invention, a secondary binding ligand is then addedto the array. Generally, the secondary binding ligand is added if theassay is an indirect assay such as a “sandwich assay” (when the targetanalyte is not an enzyme), as described in further detail herein. Thesecondary binding ligand, as discussed above, will associate with orbind to the bound target analyte and comprises an enzymatic component.The secondary binding ligand is added in an amount sufficient to ensurethat a ligand comes into contact with every bound target analyte in thearray. Alternatively, no secondary binding ligand is added, such as, forexample, when the target analyte is going to be detected directly.

A chromogenic enzymatic substrate as described above is then introducedor added to the array. The chromogenic enzymatic substrate is providedin an amount sufficient to contact any captured target analyte. Thechosen substrate reacts with or is modified by the enzymatic componentsuch that the reaction produces a chromogenic product and thus anoptical signal. The presence of the chromogenic product in the array canprovide information about the identity and/or concentration of ananalyte based on the interaction of the analyte with the capturecomponent and the enzymatic substrate (and the secondary binding ligand,in some cases).

In one embodiment of the present invention, the microwells are sealedafter the enzymatic substrate is added. That is, a sealing component isplaced in contact with the face of the substrate, thereby fluidlyisolating each microwell and sealing its contents therein. A “sealingcomponent,” as used herein, is defined as any material or device largeenough to cover the entire surface of the array substrate and capable ofcontacting the array substrate surface such that each reaction vessel issealed or isolated such that the contents of each vessel cannot escapethe vessel. According to one embodiment, the sealing component is asilicone elastomer gasket that is placed against the substrate surfacewith a uniform pressure across the entire substrate. By sealing thecontents in each microwell, the enzymatic reaction can proceed withinthe microwell, thereby producing a detectable amount of the chromogenicproduct that is retained in the microwell for detection purposes. Thatis, the enzyme converts the substrate into a chromogenic product thatbuilds up to a locally high concentration in each sealed vessel,generating a detectable chromogenic signal.

According to one embodiment, the present invention provides for amicroscope system equipped with a mechanical platform that applies thesealing component. The platform is positioned beneath the microscopestage on the microscopy system. After the assay contents have been addedto each well, the sealing component is sandwiched between a flat surface(such as, for example, a microscope slide) and the array substrate usinguniform pressure applied by the mechanical platform.

The assays may be run under a variety of experimental conditions, aswill be appreciated by those in the art. A variety of other reagents maybe included in the screening assays. These include reagents like salts,neutral proteins, e.g. albumin, detergents, etc which may be used tofacilitate optimal protein-protein binding and/or reduce non-specific orbackground interactions. Also, reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., may be used. The mixture ofcomponents may be added in any order that provides for the requisitebinding. Various blocking and washing steps may be utilized as is knownin the art.

The microwells exhibiting activity or changes in their optical signaturemay be identified by a conventional optical train and optical detectionsystem. Depending on the particular chromogenic enzymatic substratesused and the operative wavelengths of their chromogenic products,optical filters designed for a particular wavelengths may be employedfor optical interrogation of the microwells. In a preferred embodiment,the system or array of the present invention is used in conjunction withan optical fiber bundle or fiber optic array as a substrate.

According to one embodiment, the array of the present invention can beused in conjunction with an optical detection system such as the systemdescribed in U.S. application Ser. No. 09/816,651, which is incorporatedherein by reference in its entirety. For example, according to oneembodiment, the array of the present invention is the distal end of afiber optic assembly comprising a fiber optic bundle constructed of cladfibers so that light does not mix between fibers. As depicted in the09/816,651 application, the proximal end of the bundle is received by az-translation stage and x-y micropositioner.

The optical detection system of U.S. application Ser. No. 09/816,651operates as follows. Light returning from the distal end of the bundleis passed by the attachment to a magnification changer which enablesadjustment of the image size of the fiber's proximal or distal end.Light passing through the magnification changer is then shuttered andfiltered by a second wheel. The light is then imaged on a charge coupleddevice (CCD) camera. A computer executes imaging processing software toprocess the information from the CCD camera and also possibly controlthe first and second shutter and filter wheels.

The array or system of the present invention may be attached to thedistal end of the optical fiber bundle using a variety of compatibleprocesses. Wells are formed at the center of each optical fiber of thebundle. Thus, each optical fiber of the bundle conveys light from thesingle microwell formed at the center of the fiber's distal end. Thisfeature is necessary to enable the interrogation of the opticalsignature of individual microwells to identify reactions in eachmicrowell. Consequently, by imaging the end of the bundle onto the CCDarray, the optical signatures of the microwells are individuallyinterrogatable.

A. Detection

In one aspect of the present invention, the present array can be used todetect the presence of a target analyte in a sample. More specifically,the invention provides a method for detecting the product of theenzymatic reaction as an indication of the presence of the targetanalyte.

The method of detection can proceed either directly or indirectly. Ifthe target analyte is an enzyme, the analyte can be identified by adirect method of detection. Alternatively, if the target analyte is notan enzyme and thus cannot produce a chromogenic product in the presenceof a chromogenic enzymatic substrate, the analyte is identified by anindirect method of detection.

The direct method of detection, which involves a target analyte that isan enzyme, proceeds as follows. First, the sample of interest and thearray are placed in contact as described in further detail above undersuitable conditions. Subsequently, the chromogenic enzymatic substrateis added.

The presence or absence of the target analyte in any given microwell isthen detected by optical interrogation. That is, any change in theoptical signal caused by production of a chromogenic product isdetected. In any microwell containing the target analyte, the analytemodifies or acts upon the substrate in some way, thereby resulting inthe release of a chromogenic product, resulting in a change in theoptical signal from the microwell. The chromogenic reaction product isthen optically detected.

In one embodiment of the present invention, the microwells are sealedafter the enzymatic substrate is added, as described above.

The indirect methods of detection involve a target analyte that does nothave enzymatic properties. Two indirect methods that can be used withthe present invention are the “sandwich” assay and the “competitive”assay.

A sandwich assay can be performed as depicted in FIG. 2. First, thesample of interest and the array 10 are placed in contact as shown inFIG. 2 a and as described in further detail above. Under suitableconditions, target analyte 12 present in the sample is captured by thecapture components 16 in the microwells 14, as shown in FIG. 2 b.According to one embodiment, a wash step is then performed.

Next, a solution binding ligand 18 (also referred to herein as a“secondary binding ligand”) is added to the array 10, as shown in FIG. 2c. Solution binding ligands 18 are similar to capture components 16 inthat they bind to target analytes 12. The solution binding ligand 18 maybe the same or different from the capture binding ligand 16. The bindingof the solution binding ligand 18 to a captured target analyte 12 formsa “sandwich” of sorts. In the absence of the target analyte, thesolution binding ligand 18 is washed away.

A solution binding ligand 18 has two components—a binding component 22and an enzymatic label 24. The binding component 22 is the portion ofthe solution binding ligand 18 that binds to the target analyte 12.Typically, the solution binding ligand 18 binds to a different portionof the target analyte 12 than the capture component 16, because if boththe capture component 16 and solution binding ligand 18 were to bind tothe same portion, the solution binding ligand 18 would not be capable ofbinding to a captured target analyte 12. Thus, the chosen secondarybinding ligand 18 can bind to the target analyte 12 while the targetanalyte 12 is bound to a microwell 14 via a capture component 16.

The enzymatic label 24 is the portion of the solution binding ligand 18that exhibits enzymatic activity. According to one embodiment, theenzymatic label 24 is an enzyme attached to the solution binding ligand18.

Subsequently, the chromogenic enzymatic substrate is added.

In one embodiment of the present invention, the microwells are sealedafter the enzymatic substrate is added, as described above.

The presence or absence of the target analyte in any given microwell isthen detected by optical interrogation. That is, any change in theoptical signal caused by production of a chromogenic product isdetected. In any microwell containing the target analyte and thesecondary binding ligand, the enzyme associated with the secondarybinding ligand modifies or acts upon the substrate in some way, therebyproducing a chromogenic product, resulting in a change in the opticalsignal from the microwell. The product is then optically detected.

The competitive assay operates as follows. First, a labelled molecule isadded to the array of the present invention, wherein the label is aenzyme or enzymatic component. In this embodiment, the chosen labelledmolecule binds with the capture component such that the addition of thelabelled molecule to the array results in labelled molecules being boundto capture components in the microwells.

Next, the sample of interest and the array are placed in contact asdescribed in further detail above. The presence of the target analyte inthe array causes the displacement of the labelled molecule and bindingof the analyte to the capture components. The displacement occurs forthe following reason: in this embodiment, the chosen capture componentis capable of binding to either of the labelled molecule or the targetanalyte, thus resulting in a competitive binding situation. As a result,if a labelled molecule is bound to a capture component in a microwelland a target analyte is added, the target analyte will displace thelabelled molecule under suitable conditions.

According to one embodiment, a wash step is then performed to remove anynon-bound labelled molecules from the array.

Subsequently, the chromogenic enzymatic substrate is added. And asdiscussed above, according to one aspect of the invention, themicrowells are sealed after the enzymatic substrate is added.Alternatively, the microwells are not sealed.

The presence or absence of the target analyte in any given microwell isthen detected by optical interrogation. But unlike the opticalinterrogations that are described above, in this interrogation it is thelack of a chromogenic product that indicates the presence of the targetanalyte in the microwell. In any microwell containing the targetanalyte, no enzymatic action occurs and no change occurs in the opticalsignal from the microwell. In contrast, in any microwell in which thelabelled molecule is still present, an optical signal is detected.

In an alternative version of the competitive assay embodiment, both thelabelled molecule and sample of interest are added to the array at thesame time in fixed volumes. In this version, the target analyte andlabelled molecule compete directly for the binding sites on the capturecomponents.

1. Subpopulations of Identical Capture Components to Same Target Analyte

In accordance with one detection embodiment, sensor redundancy is used.In this embodiment, a plurality of reaction vessels comprising identicalcapture components referred to as “subpopulations” are used. That is,each subpopulation comprises a plurality of identical capture componentspresent in microwells of the array. Further, according to oneembodiment, each subpopulation comprises a plurality of microwellscomprising identical capture components. By using a number of identicalcapture components for a given array, the optical signal from eachmicrowell can be combined for the subpopulation and any number ofstatistical analyses run, as outlined below. This can be done for avariety of reasons. For example, in time varying measurements,redundancy can significantly reduce the noise in the system. Fornon-time based measurements, redundancy can significantly increase theconfidence of the data.

The number of subpopulations, according to one embodiment, can rangefrom 2 to any number of subpopulations possible given the limitations ofany known array and the number of different capture components.Alternatively, the number can range from about 2 to about 10. In afurther alternative, the number can range from about 2 to about 5.

In one embodiment, a plurality of identical capture components are used.As will be appreciated by those in the art, the number of identicalcapture components in a subpopulation will vary with the application anduse of the sensor array. In general, anywhere from 2 to thousands ofidentical capture components may be used in a given subpopulation, withfrom 2 to 100 being preferred, 2 to 50 being particularly preferred andfrom 5 to 20 being especially preferred. In general, preliminary resultsindicate that roughly 10 identical capture components in a subpopulationgives a sufficient advantage, although for some applications, moreidentical capture components can be used.

Once obtained, the optical response signals from a plurality ofmicrowells within each subpopulation (that is, having the same capturecomponent) can be manipulated and analyzed in a wide variety of ways,including baseline adjustment, averaging, standard deviation analysis,distribution and cluster analysis, confidence interval analysis, meantesting, etc.

2. Multiple Different Capture Components to Same Target Analyte

In addition to the sensor redundancy, the array of the present inventionaccording to one embodiment utilizes a plurality of capture componentsthat are directed to a single target analyte but are not identical. Thisembodiment provides for more than one different capture component ineach microwell or different capture components in different microwells.In one example, a single target analyte may be provided to which two ormore capture components are capable of binding. This adds a level ofconfidence as non-specific binding interactions can be statisticallyminimized. In this embodiment, when proteinaceous target analytes are tobe evaluated, preferred embodiments utilize capture components that bindto different parts of the target. For example, when two or moreantibodies (or antibody fragments) to different portions of the sametarget protein are used as capture components, preferred embodimentsutilize antibodies to different epitopes. Similarly, when nucleic acidtarget analytes are to be evaluated, the redundant nucleic acid probesmay be overlapping, adjacent, or spatially separated. However, it ispreferred that two probes do not compete for a single binding site, soadjacent or separated probes are preferred.

In this embodiment, a plurality of different capture components may beused, with from about 2 to about 20 being preferred, and from about 2 toabout 10 being especially preferred, and from 2 to about 5 beingparticularly preferred, including 2, 3, 4 or 5. However, as above, moremay also be used, depending on the application.

3. Multiple Different Capture Components to Multiple Target Analytes

According to another embodiment, the array of the present inventionutilizes a plurality of different capture components that are directedto a plurality of target analytes. This embodiment includes more thanone different capture component in each microwell or different capturecomponents in different microwells. In one example, two or more targetanalytes may be provided to which two or more capture components in thesame microwells or different microwells are capable of binding.

In this embodiment, more than one target analyte can be identified. Forexample, two or more target analytes can be identified so long as eachdifferent analyte is a different enzyme or has a different enzymaticcomponent such as a enzymatic surface molecule. In one embodiment, thetarget analytes are identified using multiple enzymatic substrateswherein each substrate produces a different color upon interaction withthe appropriate enzyme. Thus, each target analyte can be distinguishedbased on the color produced by reaction with the substrate. In analternative embodiment, the target analytes are identified usingmultiple substrates that each produce the same color. Thus, each targetanalyte can be distinguished by added the substrates sequentially.

In this embodiment, a plurality of different capture components may beused, with from about 2 to about 20 being preferred, and from about 2 toabout 10 being especially preferred, and from 2 to about 5 beingparticularly preferred, including 2, 3, 4 or 5. However, as above, moremay also be used, depending on the application.

Please note that each of the different assay configurations above,including the capture component subpopulations directed to differenttarget analytes and the plurality of capture components directed to thesame analyte, can also be utilized for quantification as describedbelow.

B. Quantification

According to one embodiment of the present invention, the present arraycannot only be used for detection of a target analyte in a sample, butalso for quantification of the analyte in the sample. That is, there isa correlation between the percentage of reaction vessels containingtarget analytes and the concentration of the analyte in the sample.Thus, the quantification method of the present invention allows forcalculation of the amount of a target analyte in a sample based on thepercentage of microwells that captured a target analyte.

Without being limited by theory, the quantification method is driven inpart by the fact that the number and volume of reaction vessels employedgovern the dynamic range of concentrations that can be determined bythis technique. That is, based on the number and volume of the reactionvessels in an array of the present invention, an estimate can be made ofthe range of concentrations of target analyte in solution that allow forthe concentration to be determined using the method of the presentinvention.

For example, for an array as disclosed in Example 2 with reactionvessels each having a volume of 46 fL, a solution having a concentrationof 3.6×10⁻¹¹ M β-galactosidase will yield, on average, one enzymemolecule per vessel. However, it is important to note that distributinga solution having a target analyte concentration within the appropriaterange into an array of reaction vessels will not result in thedistribution of exactly one enzyme molecule per vessel; statistically,some vessels will have multiple molecules while others will have zero.In the case where the number of enzyme molecules per vessel is high, thedata can be fit to a Gaussian distribution. As the ratio of enzymemolecules to reaction vessels approaches zero, the Poisson distributionapplies. This limiting distribution is used to calculate the probabilityof rare events occurring in a large number of trials. For example, basedon Poisson statistics, for a concentration of 3.6×10⁻¹¹ M, adistribution between zero and five enzyme molecules per container isobserved, with the most probable values being zero and one.

Equation 1 can be used to determine the probability of observing vevents based on the expected average number of events per trial, μ.P _(μ)(v)=e ^(−μ)(μ^(v) /v!)  Equation 1:

If the concentrations used are much less than 3.6×10⁻¹¹ M, the expectedaverage becomes exceptionally low, the distribution is narrowed, and theprobability of observing anything other than 0 or 1 events per trial isimprobable in all experimental cases. At these low concentrations, therelationship between the percentage of active reaction vessels and thebulk enzyme concentration is approximately linear. Thus, based on thisknowledge, the array of the present invention can be used to determinethe concentration of a target analyte in a sample by a simple digitalreadout system as described herein.

According to one embodiment, the quantification method of the presentinvention can be performed as follows. The method is a digital readoutsystem (also referred to as a “binary readout system”) that includesfirst detecting the target analytes in the array of microwells by anydetection method described above. The number of reaction vessels is thencounted and a percentage of the total number of reaction vessels iscalculated. That is, utilization of a yes or no response, in conjunctionwith the high-density array of reaction vessels, permits the digitalreadout of bulk concentrations of β-galactosidase. This readout isaccomplished by counting the vessels containing an active enzymemolecule across the array, with the resulting “active well” percentagecorrelating to the enzyme concentration. Given the large number ofvessels simultaneously interrogated in the array of the presentinvention, the ratio of enzyme molecules to reaction vessels could be aslow as 1:500, as the large number of wells provides a statisticallysignificant signal even at this low ratio.

Without being limited by theory, it is believed that the quantificationmethod of the present invention is only limited by the number ofindividual reaction vessels that can be viewed with an acceptableresolution. Thus, expanding the number of vessels that are interrogatedby using higher density CCD chips will decrease the limit of detectionas the lower limit is defined by the statistics of the small number ofactive wells that light up at the lower target concentrations. On theother hand, the upper limit of the dynamic range is controlled by thewell-to-well deviation from a binary readout. As target concentrationsare increased, the binary readout is lost, as a Gaussian distributionbecomes a better approximation of target molecule binding. Higherconcentrations of target lead to a broad distribution in the number ofenzyme molecules that can occupy each well, and consequently, thetransition to a non-linear increase in the percentage of active wells.

The limitations of this technique are realized above and below thethresholds of the dynamic range. As the concentration goes below thelower limit of the dynamic range, the number of enzyme molecules is toolow to observe sufficient occupied wells and, therefore, the number ofwells must be increased in order to make sure that a statisticallysignificant number of them are occupied by enzyme molecules. Results forextremely dilute concentrations have large relative errors associatedwith them, due to the very small number of reaction vessels that areexpected to show activity. Slight deviation from the expected Poissonvalue, in this case, will result in a large error. The ultimate upperlimit to this technique occurs when 100% of the reaction vessels containat least one enzyme molecule. At this limit, discrimination between twosolutions of high enzyme concentrations is not feasible. As thepercentage of active vessels approaches 100%, the linearity betweenconcentration and active vessel percentage is lost. This situationresults in a broadening distribution, as a normal distribution becomesan increasingly better approximation of the results.

In one aspect of the present invention, the array can also be used toanalyze enzyme kinetics. “Enzyme kinetics” as used herein refers to thestudy of the rates of enzyme-controlled reactions. It is understood inthe art of enzyme kinetics that the rate of an enzymatic reaction at lowsubstrate concentrations is proportional to the substrate concentration(is “substrate dependent”). This is referred to as first order. It isfurther understood that the rate of the reaction at high substrateconcentrations reaches a maximum rate and is independent of substrateconcentration because the reaction becomes saturated. Thus, if reactionvelocity is plotted as a function of substrate concentration, the lineinitially increases linearly with an increase in substrate and thenbegins to level off as substrate concentration approaches saturation.

Thus, according to one embodiment, the kinetics of any particular enzymecan be studied using the present system and array. Reaction velocityvaries across enzymes for various reasons, including, for example,reaction inhibition caused by allosteric inhibition. The array of thepresent invention allows for study of these varied kineticcharacteristics.

According to one embodiment, kinetics are examined in the followingfashion. The target analyte is allowed to bind to the capture component,the substrate is added, and the reaction vessel is sealed. Given that afinite amount of substrate is present in the reaction vessel and that nofurther substrate can be added due to the sealing of the vessel, thereaction velocity can be determined based on the amount of chromogenicproduct detected over time.

VI. Exemplary Uses of the Present Invention

The system and array of the present invention has many uses. Forexample, the array has application to fundamental enzymology studies, aswell as digital concentration measurements. Further, the array permitsstudies with multiple different enzymes and extends the limits ofultra-low detection for protein and DNA targets. With the ability tosimultaneously monitor a large array of reaction vessels, singlemolecule enzymology can be used to resolve individual enzyme moleculebehavior from bulk kinetic signal.

Another use, for example, is environmental monitoring of bacteria orviruses or both. An environmental sample potentially containing certainbacteria can be placed in contact with an array of the presentinvention. To detect the bacteria, the bacteria cells are lysed and abacterial enzyme (or more than one enzyme) is targeted for detection.According to one embodiment, the cells are lysed prior to being added tothe array. Alternatively, the cells are captured and a lysing stepoccurs on the array prior to detection. In a further alternative, nolysis may be necessary if a cell surface marker is targeted. Forexample, the bacteria or virus of interest can be captured with anantibody that is specific to a surface marker on the target, and thenthe capture can be detected with a sandwich-type assay by adding anenzyme-labelled antibody that binds to the target in another location.

Please note that all references disclosed herein are incorporated hereinby reference in their entirety.

Although the present invention has been described herein with referenceto preferred embodiments, persons skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

EXAMPLES Example 1

In this example, a proof-of-concept binding assay is performed usingenzymatic signal amplification in an array of femtoliter sized reactionvessels. More specifically, various assays are performed to detectvarying amounts of streptavidin-β-galactosidase (SβG) in solution usinga biotinylated array of the present invention and then the correlationbetween the number of wells with captured SβG molecules and theconcentration of the SβG in the sample is examined.

In this example, an etched fiber optic array is used to create acollection of femtoliter sized reaction vessels, each specificallyfunctionalized and capable of capturing enzyme-labeled target molecules.Single enzyme molecules are confined to individual reaction vessels andcatalyze the production of a sufficient number of fluorescent productmolecules to generate a positive signal. At low target moleculeconcentrations, only a percentage of the capture sites bind a targetmolecule, enabling a binary readout of target concentration from thehigh-density array.

Materials

The reactor vessel arrays in this example are generated using an acidetch of the distal face of a polished 1 mm fiber optic array, consistingof 24,000 individual 4.5 μm optical fibers. The core fiber material issilica, and the cladding around each fiber is germania-doped silica,which etches at a slower rate. The 4.5 μm fibers are etched to a depthof 2.9 μm, creating an array of reactor vessels, each with a 46 fLvolume (see FIG. 1 a).

The fibers were first modified with an aminopropyl silane bound to boththe core and cladding surfaces (see FIG. 1 b). To avoid biotinattachment to the cladding, the amino-silanized fibers were polished for10 seconds with 0.3 μm lapping film, which removed the topamino-silanized cladding layer from the fiber array (see FIG. 1 c).After polishing, NHS-biotin was attached to the amino groups on the wellsurfaces (see FIG. 1 d).

Methods

First, the effectiveness of the capture component was tested. To testthe effectiveness of the biotinylation of the substrate, streptavidinAlexa Fluor 568® was attached directly to the biotin groups on thesurfaces of both a polished and an unpolished fiber, followed by imageacquisition of the modified surface (see FIG. 3). FIG. 3 showsStreptavidin Alexa Fluor 568® binding to (a) an unpolished biotinmodified fiber optic array, and (b) a polished biotin modified fiberoptic array. As seen in image (a), streptavidin binding occurred on allsurfaces, in comparison to image (b), where binding occurred only on thesurfaces of the microwell reactors. Thus, the unpolished fiber shows dyeover the entire array including the cladding surface, while the polishedfiber shows dye localized only on the well surfaces.

Subsequent to array modification, the biotinylated fiber arrays wereincubated for 1 hour at room temperature in 150 μL PBS buffer containingvarying amounts of SβG. The concentration of the SβG was chosen so thatduring the incubation time, statistically either one molecule or nomolecules would bind to each well. The arrays were then washedrepeatedly in PBS buffer, to ensure that unbound target was removed.

For a binary readout of SβG binding, the fiber array was loaded andsecured on an upright microscope system equipped with a mechanicalplatform. A solution of β-galactosidase substrate,resorufin-β-D-galactopyranoside (RDG), was introduced to the distal endof the fiber containing the reaction vessels, and subsequently sealed.The substrate was sealed using a 0.01-inch thick silicone elastomergasket sandwiched between a microscope slide and the fiber array bymeans of a mechanical platform located beneath the microscope stage.This platform applied a uniform pressure to the gasket material, acrossthe entire bundle, sealing off each reaction chamber and enabling wellto well interrogation of enzyme activity. β-galactosidase hydrolyzes RDGto form resorufin, which builds up to a locally high concentration ineach sealed reaction vessel, generating a detectable fluorescent signal(FIG. 4).

FIG. 4 depicts a portion of the fiber array for each experiment. Each ofthe experiments tested a different sample having a differentconcentration of SβG. The concentrations for each experiment were asfollows: (a) 128 amol, (b) 51 amol, (c) 25 amol, (d) 7.5 amol, and (e)2.6 amol. FIG. 4(f) depicts the control.

Analysis of over 5000 reaction vessels for each experiment allowed for acorrelation between the percentage of reaction vessels that captured anenzyme molecule and the amount of enzyme present in the interrogatedsample. The variation seen in the intensity differences from active wellto active well is most likely a result of molecule-to-molecule variationin catalytic activity, in combination with surface effects, which maymodulate the relative activities of enzyme molecules based on theirorientation to the reaction chamber surface.

Two control experiments were also conducted to ensure that the bindingof enzyme to the surface of the reactors was based exclusively on thebiotin-streptavidin interaction, and not on non-specific binding to theglass surface. One control experiment consisted of an etched, unmodifiedfiber incubated with the most concentrated SAG target solution (128 amolin 150 μL). The second control experiment was performed using themodified fiber incubated in a solution of β-galactosidase lackingstreptavidin (128 amol in 150 μL). Both control experiments generated anegligible active well percentage (less than 0.06%, versus 0.2% for the2.6 amol experiment discussed below).

Results

FIG. 5 depicts a log-log plot of the moles of target present in a samplewith the resulting percentage of active reaction vessels. The linearrelationship between the percentage of active reaction vessels and themoles of target in the log-log plot shown in FIG. 5 suggests that abinary readout detection method can be used for the detection of realtargets such as DNA and antigens. This method permits rapid analysis andaccurate concentration information via digital readout, whilemaintaining a straightforward assay procedure.

It is also interesting to note that the lowest limit of detection (LOD)for binding streptavidin-β-galactosidase (SβG) to a biotinylatedfemtoliter array in this example was 2.6 amoles (150 μL of 17 fMsolution) using a target incubation time of 1 hour.

Example 2

In this example, single molecules of β-galactosidase were monitoredusing a 1 mm diameter fiber optic bundle with over 2.0×10⁵ individuallysealed, femtoliter microwell reactors. By observing the buildup offluorescent products from single enzyme molecule catalysis over thearray of reaction vessels and by applying a Poisson statisticalanalysis, a digital concentration readout was obtained.

Materials

1 mm bundled 4.5 μm optical fibers were purchased from Illumina (SanDiego, Calif.). β-galactosidase and Ru(bpy)₃Cl₂ was obtained fromSigma-Aldrich (St. Louis, Mo.). Resorufin-D-β-galactopyranoside waspurchased from Molecular Probes (Eugene, Oreg.). 0.01-inchnon-reinforced gloss silicone sheeting material was purchased fromSpecialty Manufacturing Inc. (Saginaw, Mich.). All other chemicals usedwere of reagent grade and obtained from Sigma-Aldrich (St. Louis, Mo.).

A custom-built, upright epifluorescence imaging system acquired allfluorescence images using a mercury light source, excitation andemission filter wheels, microscope objectives, and a CCD camera (QE,Sensicam). Filter wheels and shutters were computer controlled andanalysis was performed with IPlab software (Scanalytics, Fairfax, Va.).The system was equipped with a fastening device to fix the fiber opticarray onto the system through the entire experiment. A mechanicalplatform beneath the stage was used to house the silicone-sealing layer,which was subsequently brought into contact with the distal end of thefiber array, sealing off each reaction vessel. All measurements wereperformed with femtowell arrays at the distal end of the optical fiberbundle.

Optical fiber bundles containing approximately 2.4×10⁵ individual 4.5 μmdiameter optical fibers were used as the substrate for fabricatingfemtoliter reaction vessel arrays. The well volume can be preciselycontrolled, as etch depth varies with etch time and etchantconcentration. The optical fibers used in these experiments were etchedto a depth of approximately 2.9 μm, yielding a 46 μL well volume. FIG. 6depicts images of the etched surface of the fiber optic bundles. Morespecifically, FIG. 6 a depicts the entire fiber array and close-upmicroscope images of the fiber bundle, emphasizing the regularity ofboth the array and each individual optical fiber. Further, FIG. 6 b isan AFM image of a portion of the etched surface, showing wells createdfrom the etching process.

Methods

Assay. For the β-galactosidase assay, the substrate used wasresorufin-β-D-galactopyranoside. After the individual wells in the arraywere sealed in the presence of enzyme and substrate, the fluorescenceintensity was monitored across the array of vessels for the enzymaticproduct, resorufin (ex 558 nm/em 573 nm). A 100 μM solution ofresorufin-D-β-galactopyranoside (RDG) was prepared in 100 mM Tris bufferpH 8.0 containing 2.0 mM KCl and 0.1 mM MgCl₂. All enzyme solutions wereprepared from previously aliquoted and frozen stock samples in the samereaction buffer. Just prior to experimentation, the two samples werecentrifuged for 2 min at 7000 RPM to remove any particulate materialthat could interfere with the mechanics of the silicone seal.Approximately 1 cm² of silicone and a microscope slide were cleaned withabsolute ethanol. The silicone sheeting was placed on the surface of theglass, to which it adhered. Subsequently, 75 μL volumes of enzyme andRDG solutions were mixed on the silicone gasket using a pipette. Thegasket was mechanically raised towards the distal end of the fiberbundle until it experienced resistance, suggesting that a seal wasformed. An initial fluorescence image was acquired, followed by periodicimage acquisition for approximately 2 hr.

Sealing component. To seal the femtoliter array, a 0.01-inch thicksilicone elastomer gasket was sandwiched between a microscope slide andthe fiber array using a mechanical platform. This platform applieduniform pressure to the gasket material, across the entire bundle,sealing off each microwell to create the reaction vessels.

The silicone/glass seal used to create and isolate the femtolitercontainers was inspected for its sealing ability by performing aphotobleaching experiment (see FIG. 7). FIG. 7 depicts enclosure of asolution into the microchambers and evaluation of the silicone seal forintegrity. FIG. 7 a depicts a solution of Ru(bpy)₃Cl₂ enclosed into thearray of chambers as observed by the red fluorescence across the array.FIG. 7 b depicts a small octagonal portion of the fiber bundle that wasphotobleached via UV light. FIG. 7 c depicts the array 60 minutes later.As shown in the figure, diffusion of Ru(bpy)₃Cl₂ from one well toanother as a result of an imperfect silicone seal would displayincreased fluorescence intensity in photobleached wells and was notobserved. This experiment substantiated the integrity of the seal forits ability to successfully isolate the array of vessels. Enzymemolecule denaturation on the glass surface was prevented by blockingwith a BSA blocking buffer. Enzyme to vessel ratios used ranged from1:5, down to 1:500, achieving accurate detection over two orders ofmagnitude.

Photobleaching Experiment. A solution of 1 mM Ru(bpy)₃Cl₂ in DI waterwas used for the photobleaching experiments. A piece of silicone,approximately 1 cm², and a microscope slide were cleaned with absoluteethanol using lint-free swabs. The silicone sheeting was placed on thesurface of the glass, to which it adhered. 50 μL of the Ru(bpy)₃Cl₂solution was placed on the silicone, and subsequently brought intocontact with the fiber bundle, to enclose the solution in the individualvessels. Using a field stop on the imaging system, UV light was used toilluminate a small portion of the array for 10 minutes, photobleachingthe Ru(bpy)₃Cl₂. The field stop was then opened, and an image wasacquired, displaying the difference in fluorescence. The array was thenallowed to rest with the seal maintained. A final image was taken after60 minutes, confirming the integrity of the seal.

As discussed above, the number and volume of reaction vessels employedgovern the dynamic range of concentrations that can be determined bythis technique. The reaction vessel volumes employed in this examplewere 46 fL (vide infra); therefore, it was calculated that a solution of3.6×10⁻¹¹ M β-galactosidase will yield, on average, one enzyme moleculeper vessel. As also discussed above, if the concentrations used are muchless than 3.6×10⁻¹¹ M, the expected average becomes exceptionally low,the distribution is narrowed, and the probability of observing anythingother than 0 or 1 events per trial is improbable in all experimentalcases. At these low concentrations, the relationship between thepercentage of active reaction vessels and the bulk enzyme concentrationis approximately linear. After waiting for sufficient time to allowenzyme catalysis to occur, individual vessels were interrogated for anon/off response, correlating to each vessel either possessing or lackingenzymatic activity.

The substrate resorufin-D-β-galactopyranoside (RDG) was used as thesubstrate for experiments, which was sealed into all the vessels, alongwith the trapped enzyme molecules, using a silicone gasket material andmechanical arm. The expected percentages of active wells were calculatedfor each concentration used by applying the Poisson distributionstatistics.

Results

As shown in FIG. 8, for the β-galactosidase assay, different bulksolution enzyme concentrations correspond to different ratios of enzymeto vessel volume, resulting in variation in the percentage of vesselsthat contain an enzyme molecule. FIG. 8 depicts the detection of theactivity of single molecules of β-galactosidase. FIG. 8 a is abackground image of a portion of the array, while FIG. 8 b depicts animage taken of a portion of a 1:5 enzyme to vessel assay, and FIG. 8 cshows a 1:80 enzyme to vessel assay.

Table 1 is a comparison of each experimental result with the percentageof occupied vessels calculated from the Poisson distribution. As shownby the data in the table, the array measurements successfully correlatedwith the number of single enzyme β-galactosidase molecules over theentire range of interrogated concentrations. There is minor disparity inthe observed signals as a result of molecule-to-molecule variation incatalytic activity. This result is most likely due to the inherentstochastic nature of enzymes, in addition to surface effects, resultingin modulation of enzyme activity. TABLE 1 Digital Readout of EnzymeConcentrations Digital readout from the arrays. The actual percentage ofchambers exhibiting activity, in comparison to the expected percentagecalculated from the Poisson distribution, are listed for the variousconcentrations analyzed. Enzyme to well ratio Concentration Poisson % ofactive wells Actual % active 1:5  7.20E−12 18.2 14.9 1:10 3.60E−12 9.511.5 1:20 1.80E−12 4.9 5.6 1:40 9.00E−13 2.5 3.5 1:80 4.50E−13 1.2 1.5 1:100 3.60E−13 1.0 1.1  1:200 1.80E−13 0.5 0.3  1:500 7.20E−14 0.2 0.1

The variation between the calculated and experimental results can beattributed to the intrinsic variability associated with the probabilitydistribution, as well as experimental error in the preparation of enzymesolutions.

BIBLIOGRAPHY

Each of the following references is incorporated by reference in theirentirety.

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1. A method of detecting a reaction component that affects a reaction,the method comprising: a) providing a reaction component, a sample andan array, said sample comprising a target analyte in a firstconcentration, said array comprising at least 1000 sites, each sitehaving a defined volume between 10 attoliters and 50 picoliters; b)contacting said array with said sample such that the ratio of targetanalyte to each site of said array is less than 1:1; c) introducing saidreaction component to each site of said array; and d) determiningwhether the reaction component affects a reaction.
 2. The method ofclaim 1, wherein said target analyte is an enzyme and substrate for saidenzyme is present in each site of said array prior to step d).
 3. Themethod of claim 2, wherein said reaction component is an enzymeinhibitor.
 4. The method of claim 3, wherein said determining of step d)comprises determining whether said enzyme inhibitor inhibits thereaction of said enzyme with said substrate.
 5. The method of claim 4,wherein said substrate is a chromogenic substrate.
 6. The method ofclaim 1, wherein said ratio is less than 1:5.
 7. The method of claim 1,wherein said ratio is 1:10.
 8. The method of claim 1, wherein said ratiois between 1:5 and 1:500
 9. The method of claim 1, further comprising,after step c), sealing the array such that the contents of each sitecannot escape said site.
 10. The method of claim 1, wherein said definedvolume is measured in femtoliters
 11. The method of claim 10, whereinsaid defined femtoliter volume is the same at each site and ranges fromabout 30 femtoliters to about 60 femtoliters.
 12. The method of claim11, wherein said defined femtoliter volume is 46 femtoliters.
 13. Themethod of claim 1, wherein said array comprises between 20,000 and30,000 sites.
 14. The method of claim 1, wherein said array comprisesbetween 100,000 and 10,000,000 sites.
 15. The method of claim 1, whereineach site of said array comprises a capture component.
 16. A method ofdetecting a reaction component that affects a reaction, the methodcomprising: a) providing a reaction component, a sample and an array,said sample comprising a biomolecule target analyte in a firstconcentration, said array comprising at least 1000 sites, each sitehaving a defined volume between 10 attoliters and 50 picoliters; b)diluting said sample to create a diluted sample, said diluted samplecomprising a biomolecule target analyte in a second concentration; c)contacting said array with said diluted sample such that the ratio ofbiomolecule target analyte to each site of said array is less than 1:1;d) introducing said reaction component to each site of said array; ande) determining whether the reaction component affects a reaction. 17.The method of claim 16, wherein said biomolecule target analyte is anenzyme and substrate for said enzyme is present in each site of saidarray prior to step e).
 18. The method of claim 17, wherein saidreaction component is an enzyme inhibitor.
 19. The method of claim 18,wherein said determining of step e) comprises determining whether saidenzyme inhibitor inhibits the reaction of said enzyme with saidsubstrate.
 20. The method of claim 19, wherein said substrate is achromogenic substrate.
 21. The method of claim 16, wherein said ratio isless than 1:5.
 22. The method of claim 16, wherein said ratio is 1:10.23. The method of claim 16, wherein said ratio is between 1:5 and 1:50024. The method of claim 16, further comprising, after step d), sealingthe array such that the contents of each site cannot escape said site.25. The method of claim 16, wherein said defined volume is measured infemtoliters
 26. The method of claim 25, wherein said defined femtolitervolume is the same at each site and ranges from about 30 femtoliters toabout 60 femtoliters.
 27. The method of claim 26, wherein said definedfemtoliter volume is 46 femtoliters.
 28. The method of claim 16, whereinsaid array comprises between 20,000 and 30,000 sites.
 29. The method ofclaim 16, wherein said array comprises between 100,000 and 10,000,000sites.
 30. The method of claim 16, wherein each site of said arraycomprises a capture component.
 31. The method of claim 30, wherein saidcapture component immobilizes said enzyme.
 32. A method of detectingenzyme inhibition, the method comprising: a) providing i) a reactioncomponent, ii) a sample, said sample comprising a enzymatic targetanalyte in a first concentration, iii) a substrate for said enzymatictarget analyte, and iv) an array, said array comprising at least 10,000sites, each site having a defined femtoliter volume; b) diluting saidsample to create a diluted sample, said diluted sample comprising aenzymatic target analyte in a second concentration; c) contacting saidarray, in any order, with said substrate, said reaction component, andsaid diluted sample such that the ratio of enzymatic target analyte toeach site of said array is between 1:5 and 1:500; and d) determiningwhether the reaction component inhibits the reaction of said enzyme withsaid substrate.
 33. The method of claim 32, wherein said substrate is achromogenic substrate.
 34. The method of claim 32, further comprising,after step c), sealing the array such that the contents of each sitecannot escape said site.
 35. The method of claim 32, wherein saiddefined femtoliter volume is the same at each site and ranges from about30 femtoliters to about 60 femtoliters.
 36. The method of claim 35,wherein said defined femtoliter volume is 46 femtoliters.
 37. The methodof claim 32, wherein said ratio is 1:10.
 38. The method of claim 32,wherein said array comprises between 20,000 and 30,000 sites.
 39. Themethod of claim 32, wherein said array comprises between 100,000 and10,000,000 sites.
 40. The method of claim 32, wherein said substrate isintroduced first into each site of said array.
 41. The method of claim40, wherein said substrate is immobilized in said site as a capturecomponent.